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Cuban Journal of Agricultural Science

versión impresa ISSN 0864-0408versión On-line ISSN 2079-3480

Cuban J. Agric. Sci. vol.52 no.3 Mayabeque jul.-set. 2018  Epub 01-Sep-2018


Animal Science

Nutritive value and in vitro caecal fermentation of forage meal of mulberry variety YU 62 for rabbits

G.E. Vasallo1  * 

Lourdes Savón1 

L.E. Dihigo1 

J.G. Cairo1 

1Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Provincia Mayabeque, Cuba


The objective of this study was to determine the nutritional value and in vitro caecal fermentation in mulberry forage meal, variety YU 62, for rabbits. A completely random design was used, analyzing six times the samples of mulberry variety YU 62 forage meal with Medicago sativa (lucerne) as control. An in vitro simulation of digestive process in anatomical segments of stomach, duodenal and caecal gastrointestinal tract of rabbit was made. In stomach phase, high calcium content in mulberry raised the pH. In the duodenal phase, a lower pH with a suitable acid-base balance was obtained, which produced an increase in digestibility of crude protein (P <0.0001) in mulberry forage meal with value of 54.92% vs 36.4% respectively of lucerne control. In the caecal phase, in mulberry meal, digestibility of dry matter was higher than the values found in lucerne influenced by the higher digestibility for NDF. This is due to its high content of hemicellulose in the cecum and has a favorable effect on the activity of the caecal microbiota that coincides with the increase of short-chain fatty acid mainly acetic and butyric with significant difference (P <0.0001) with lucerne. In addition, it should be noted that increasing short chain fatty acids lowers pH, tending to neutrality. It is suggested that mulberry forage meal variety YU 62 has a positive in vitro nutritive value, so that it could be used instead of lucerne as an alternative raw material in the formulation of diets for rabbit feeding.

Key words: rabbit; mulberry; in vitro nutritional value

The development of animal production systems, currently, demand to be more profitable. One of the alternatives to achieve this is the use of trees and shrubs of high nutritional quality and performance that are fibre sources, which may be key components in rabbit feeding, because they are involved in regulating digesta passage rate through the gastrointestinal tract and conducive to proper digestive function (Rebours et al. 2017).

Mulberry tree has good nutritional characteristics and biomass production of more than 15 tDM/ ha/year. Knowledge of its digestibility is essential to propose dietary formulas that enable to establish parameters for inclusion in the diet for rabbits, and the use of the in vitro method allows to predict rapid and economically the nutritional value of feed and especially digestibility of nutrients in concentrates (Dihigo 2007).

It is also important to specify the values of caecal fermentation (short-chain fatty acids, pH and ammonia) which are indicators that reflect the activity of the caecal ecosystem, influenced by the nutrients in the diet, one of the most important factors (Knudsen, 2014). Therefore, it is important the use of caecal content in in vitro techniques.

According to the above, the objective of this study was to determine the nutritional value and in vitro caecal fermentation in mulberry forage meal, variety YU 62, for rabbits.

Materials and Methods

The study was developed at the department of Bio-physiological Sciences, in the Instituto de Ciencia Animal, located at Carretera Central km 47 ½, San José de las Lajas, Mayabeque province.

Preparation of forage feed source. Mulberry forage (Morus alba) variety YU 62, from a hectare from Guayabal experimental unit, composed by three fields with two years of establishment in a red ferrallitic soil, was used.

In the agronomic management, chemical fertilization was applied with urea and organic-based organic matter and humus. The first cut was carried out at 12 months after sowing and later, 11 cuts up to July 2014, when the plant was obtained, after that with a cutting frequency every 50 days during the rainy season. Plant cutting was conducted at 50 cm high from the soil.

The sampling process was randomLy carried out in three fields with two samples of fresh material per field, with a weight of 1 kg each. Components of edible biomass were cut, which included leaves and stems that were homogenized and woody stems were discarded. Later, drying was performed under the sun (36 hours).

Pellets of lucerne (Medicago sativa) were purchased from the Centro Nacional para la Producción de Animales de Laboratorio (CENPALAB). Six bags of lucerne were sampled with taking 5 points per each bag until a sample collection of 2kg per bag.

All samples (lucerne and mulberry) were ground with a hammer mill of 4mm and sieved to 1mm for further laboratory analyses.

Determination of the chemical composition. Chemical composition was determined six times in samples of mulberry forage and lucerne as control. Dry matter (DM), organic matter (OM), ash and crude protein (CP) contents were determined according to the method of AOAC (2016). Cell wall fractions (NDF, ADF and ADL) were determined by Goering and Van Soest (1970). NDF and ADF were corrected for ash and performed using crucibles. Cellulose (Cel) and hemicellulose (Hec) were calculated by difference between ADF-ADL and NDF-ADF, respectively. Calcium (Ca) was analyzed by atomic absorption and phosphorous (P) according to Amaral (1972) colorimetry.

In vitro digestibility and caecal fermentation. The experiment was conducted in the laboratory of in vitro digestibility of non-ruminant species, in homogeneous conditions of local temperature at 20 °C and relative humidity of 40 %.

For simulating in vitro digestive process in the anatomical segments of stomach, duodenal and caecal gastrointestinal tract (GIT), 2 g of samples (sieved to 1mm) were weighed with six repetitions per treatment (mulberry forage meal var. YU 62 and lucerne control) and per section of GIT. Samples were placed in muslin bags of 48-micron porosity and deposited in incubation tubes of 120 mL, distributed in water baths with agitation and temperature control.

The stomach-duodenum phase was developed according to the methodology described by Ramos et al. (1992), with the use of pepsin and pancreatin enzymes. Controls were placed to correct the results.

For the stomach phase, a solution of phosphate buffer (0.1 M and pH = 6) was prepared and 25 mL were added to each tube of incubation with the sample plus 10 mL of 0.2 M HCl and were homogenized. The pH was adjusted to 2 with 1 M solution of 0.1 N hydrochloric acid and 0.1 N sodium hydroxide. Then, 1 mL of freshly prepared pepsin solution containing 25 mg/mL pepsin (porcine) was added. It was capped with a rubber stopper with valves and was randomLy inserted into a thermostated bath at 39 °C during 1.5 hours. At the end of the phase, pH was measured.

In the duodenal phase, 10 mL of a buffered phosphate solution (0.2 M, pH = 6.8) and 5 mL of a 0.6 M NaOH solution were added to each tube. The pH was adjusted to 6.8 with 1M NaCl and NaOH. Afterwards, 1 mL of freshly prepared pancreatin solution containing 100 mg/mL porcine pancreatin was added. The tubes were capped and placed in thermostated bath at 39 °C during 3.5 hours. At the end of the phase, pH was measured and the samples contained in the bags were weighed to determine CP.

In the caecal phase, it has used the previous tubes used in the stomach-duodenum phase. Freshly collected samples of caecal content were used to prepare the inoculum according to method described by Pascual et al. (2000). Nine young New Zealand White x White Semi-giant (2 kg of weight) showing a normal weight gain during the fattening period were randomLy chosen prior to slaughtering. Animals consumed lucerne commercial concentrate. After adjusting pH to 6.9, 100 mL of inoculum were added to each tube and incubated at 39 °C in bath temperature-controlled for 48 hours. Upon completion of the incubation period, pH was measured and bags were washed with cold distilled water and ethyl alcohol 90 % and placed in the oven at 60 °C for 48 hours, for subsequent estimation of DM and NDF. Digestibility of DM, CP and NDF was calculated according to Ramos (1995).

Individual SCFA concentration in the preserved samples was determined by gas chromatography, on injecting 0.5 μL, after centrifuging for 8 min the vials at 14 200 x g (Centrifuge ECEN-205, MRC Ltd., Hagsvish, Israel). A liquid-gas chromatograph DANI Master GC (DANI Instruments S.p.A. Milan, Italy) was used equipped with a capillary column DN-FFAP (length 30 m, internal diameter 0.32 mm, film thickness 0.25 μ) and a FID detector. H2 was used as carrier gas and N2 as auxiliary. Maximum temperature of the injector and detector was fixed at 200 and 250 ºC, respectively. Also total SCFA were obtained by algebraic sum of the individual SCFA determined. The quotient of the concentration of acetic and propionic acids (Ac/Pp relationship) was calculated.

Statistical analyses. Variance analyses was performed using a completely randomized design, Fisher LSD test was used to know significant differences between treatments. Analyses of power (1-β) was applied to reject or to accept the null hypothesis in the necessary cases that in this case was considered a value above 0.80. Data were analyzed according to Di Rienzo et al. (2012).

Results and Discussion

Chemical composition. Differences were observed (P <0.0001) in the nutrient content of the foods under study, with higher percentage of DM and ash in the lucerne. However, OM, Ca and P were higher in the mulberry in accordance with the results reported by López et al. (2014). In studies conducted by Martin et al. (2014), referred that factors as the organic fertilization, sun dryness of the plant and the height of cutting, influence the increase of the tenor of organic matter and minerals as calcium and phosphorus. Table 1 shows chemical composition of forage meal of mulberry (Morus alba variety Yu-62) and lucerne.

Table 1 Chemical composition (% dry basis) of mulberry meal variety YU-62 and lucerne meal. 

SEM: standard error of mean.

Mulberry forage meal presented the lowest values (P<0.0001) of CP in relation to the lucerne, in correspondence with those cited by Joromocoj (2012) where the protein content is attenuated in the integral forage and tends to be higher when leaves are used and lowest when the stems are included.

This also influenced the high percentages (P <0.0001) of the fibrous constituents (crude fiber, NDF, ADF, cellulose, hemicellulose and lignin) that were higher than lucerne, with higher emphasis for neutral detergent fiber (NDF). This can be attributed, besides to the time established for the plant with more than one year, to a cutting frequency of 50 days with little rainfall regime (García et al. 2006).

In vitro digestibility and caecal fermentation. Table 2 shows digestibility of crude protein (CP) and values in pH, according to two phases of incubation (stomach and duodenal). There were differences among treatments (P <0.0001) for the analyzed indicators. In the stomach phase, pH increased in mulberry forage meal according to Dihigo (2007), this could be caused by the high content of saponin and calcium, which reacts with the acid medium.

Table 2 In vitro digestibility (%) of CP and pH values of mulberry meal variety Yu-62 and lucerne meal. 

SEM: standard error of mean.

However, in the duodenal phase, due to the high buffering capacity of mulberry, pH was lower compared to lucerne and a suitable acid-basic balance was achieved. This agrees with results of Dihigo (2007) and, according to Michelland et al. (2010), contributed to a better enzymatic activity of pancreatin, reason why protein digestibility (P <0.0001) was higher than the values found in lucerne.

The lower digestibility of proteins in lucerne, could be due to the content of antinutritional components such as soluble tannins which form indigestible complexes with proteins and interfere with their digestibility (Legendre et al. 2017). The concentration of these secondary metabolites depends on factors such as genotype and parts of the plant as it has shown higher concentration in the seed than in the aerial portions and the latter are usually present in high proportion when the plant is cut very early or too late in the growth stage (Gawel, 2012). Authors such as De Blas et al. (2010), report concentrations of soluble tannins in lucerne of 3 to 4 %. With regard to the mulberry, researches that have been realized recount absence of tannins that precipitate proteins and content of total polyphenols from 1.05 to 2.89 % (Savón et al. 2017).

In mulberry meal, digestibility of dry matter (DM) was higher than the values found in lucerne influenced by the higher digestibility for NDF. This is due to its high content of hemicellulose in the cecum and has a favorable effect on the activity of the caecal microbiota that coincides with the increase of short-chain fatty acid (SCFA) mainly acetic and butyric (Safwat et al., 2014) (table 3), with significant difference (P <0.0001) with lucerne. In addition, it should be noted that increasing SCFA lowers pH, tending to neutrality (Cossu 2014 and Gaafar and Ayat 2014), as between the two variables, there is an inverse relationship, reflecting the strong interaction of caecal ecosystem with the feed rate, when it contains easily digestible fiber (Jacquier et al. 2013).

Table 3 Caecal in vitro digestibility (%), pH and short-chain fatty acid (mmol/L) of mulberry meal variety Yu-62 and lucerne meal. 

SEM: standard error of mean.

In mulberry forage meal, propionic/butyric ratio was low (0.59) compared to lucerne (0.96) (0.02 SE P <0.0001). Authors such as Blas (2013) and Pinzon (2014) argued that when the diet is rich in fiber, concentration of caecal propionic acid decreases with less propionic/butyric ratio of 1.

It is concluded that mulberry forage meal variety YU 62 has a positive in vitro nutritive value, so that it could be used instead of lucerne as an alternative raw material in the formulation of diets for rabbit feeding and, further studies of in vivo digestibility and metabolic profile are suggested.


Thanks to Natacha Pompa, Doremis Rosales, Yusmelis Ramos and Ibett Orta for their collaboration with laboratory tests, as well as to Lucía Sarduy and Magaly Herrera Villafranca for the accomplishment of statistical analyses.


AOAC. 2016. Official Methods of Analysis. Ass. Off. Agric. Chem. 20th ed. Washington, D.C. [ Links ]

Amaral, A. 1972. Técnicas analíticas para evaluar macronutrientes en cenizas de vainas de caña de azúcar. La Habana: Editorial Pueblo y Educación, p 35. [ Links ]

Blas, I. 2013. Utilización de piensos con lactoreemplazante en conejo de cebo. Master Thesis. Universidad Politécnica de Valencia, España. 26 pp. [ Links ]

Cossu, M. E. 2014. Algunos conceptos sobre la nutrición del conejo para carne. In: Tecnología de producción de conejos para carne. Uruguay. Editorial INIA. p. 63-79. [ Links ]

De Blas, C., Mateos, G.G. & Rebollar, P.G. 2010. Alfalfa. In: Tablas FEDNA de composición y valor nutritivo de los alimentos para la fabricación de piensos compuestos. España. 3ra edición. p.282-283. [ Links ]

Dihigo, L.E. 2007. Características físico-química de productos tropicales y su impacto en la morfofisiología digestiva del conejo. Habana. PhD Thesis. Universidad Agraria de la Habana, Cuba. [ Links ]

Di Rienzo, J., Casanoves, F., Balzarini, M., González, L., Tablada, M. & Robledo, C. 2012. InfoStat version 2012. Grupo InfoStat, FCA. Córdoba, Argentina, Universidad Nacional de Córdoba. [ Links ]

Gaafar, H.M.A. & Ayat, A. 2014. Effect of diet supplemented with pumpkin (Cucurbita moschata) and black seed (Nigella sativa) oils on performance of rabbit: 1- Growth performance, blood haematology and carcass traits of growing rabbit. Report and Opinión. 6: 52-59. [ Links ]

García, D., Noda, Y., Medina, M., Martín, G. & Soca, M. 2006. La morera: una alternativa viable para los sistemas de alimentación animal en el trópico. Avances en investigación agropecuarias. 10: 55-72. [ Links ]

Gawel, E. 2012. Chemical composition of lucerne leaf extract (EFL) and its applications as a phytobiotic in human nutrition. Acta Sci. Pol., Technol. Aliment. 11: 303-310. [ Links ]

Goering, H. K. & Van Soest, P. J. 1970. Forage Fiber Analysis (Apparatus, Reagents, Procedures, and Some Appliccations), Agric. Handbook No. 379. Ars- USDA. Washington, DC. [ Links ]

Jacquier, V., Combes, S., Oswald, I.P., Rogel-Gaillard, C. & Gidenne, T. 2013. Incorporation de fibres rapidement fermentescibles dans un aliment périsevrage: impact sur la digestion, la croissance et l´état sanitaire du lapin. 15èmes Journées de la Recherche Cunicole, 19-20 novembre 2013, Le Mans, France [ Links ]

Joromocoj, E.D. 2012. Evaluación del contenido de proteína y biomasa en la morera (Morus alba, L.; Moraceae) con la aplicación de tres fuentes orgánicas de fertilización, en sololá, Guatemala. Graduated Thesis. Universidad Rafael Landívar, Quetzaltenango, Guatemala. [ Links ]

Knudsen, C. 2014. Strategies de limitation de l´ ingestion chez le lapin: optimisation des performances zootechniques, impacts physiologiques et consequences sur la santé digestive. These de Docteur en Sciences. Université de Toulouse, France. [ Links ]

Legendre, H., Hoste, H., Gombault, P., Routier, M., Bannelier, C. & Gidenne, T. 2017. Valeur nutritive du sainfoin déshydraté, lors d´une forte substitution dans un régime á base de luzerne dans l´ alimentation du lapin en croissance. 17èmes Journées de la Recherche Cunicole, 21 et 22 novembre 2017, Le Mans, France [ Links ]

López, B., Iser, M., Cisnero, M., Ramírez, J.L., Valdivié, M. & Savón, L. 2014. Inclusión de la harina de Morera (Morus alba) en el desempeño productivo de conejos. Revista de producción animal 26: 2-8. [ Links ]

Martin, G.J., Pentón, G., Noda, Y., Contino, Y., Díaz, M., Ojeda, F., Jiménez, F.A., López, O., Agramonte, D., Milera, M. & Prieto, M. 2014. Comportamiento de la morera (Morus alba L.) y su impacto en la producción animal y la crianza de gusanos de seda en Cuba. Revista Cubana de Ciencia Agrícola 48:73-77. [ Links ]

Michelland, R.J., Combes, S., Monteils, V., Cauquil, L., Gidenne, T., Fortun-Lamothe, L., 2010. Molecular analysis of the bacterial community in digestive tract of rabbit. Anaerobe, 16, 61-65. [ Links ]

Pascual, J., Fernández-Carmona, J., Ferández, Díaz, J. R., Garcés, C., Rubert-Alemán, J., Llopis & Muelas, R. 2000. Nutritive evaluation of rabbit diets by different in vitro digestibility Methods. J of World Rabbit Sci Association. 8: 385-389. [ Links ]

Pinzon, O.F. 2014. Evaluación del efecto del uso de bloques multinutricionales basados en morera sobre los parámetros productivos de conejos Nueva Zelanda. Departamento de Boyacá. Trabajo presentado como requisito parcial para optar por el título de Zootecnista-Universidad Nacional Abierta y a Distancia, Colombia. 78pp. [ Links ]

Ramos, M., Carabaño, R & Boisen. 1992. An in vitro methods for estimating digestibility in Rabbit. J. Appl. Rabbit Res. 15: 938-946. [ Links ]

Ramos, M.A. 1995. Aplicación de técnicas enzimáticas de digestión in vitro a la valoración nutritiva de piensos en conejo. Madrid. PhD Thesis. Universidad Complutense de Madrid. [ Links ]

Rebours, G., Vastel, P., Bouchier, M., Faussier, G. & Reys, S. 2017. Comparaison de deux méthodes de dosage de la fraction de ligneuse sur des matiéres premiéres fibreuses: impact sur le risque de trouble digestifs chez le lapin en croissance. 17èmes Journées de la Recherche Cunicole , 21 et 22 novembre 2017, Le Mans, France [ Links ]

Savón, L., Gutierrez, O. & Febles, G. 2017. Mulberry, moringa and tithonia in animal feed, and other uses. Results in Latin America and the Caribbean. La Habana. EDICA. 280p. ISBN: 978-959-7171-72-0. [ Links ]

Safwat, M.A., Sarmiento-Franco, L. & Santos-Ricalde, R.H. 2014. Rabbit Production using local resources as feedstuffs in the tropics. Tropical and Subtropical Agroecosystems. 17: 161-171. [ Links ]

Received: February 05, 2018; Accepted: August 06, 2018

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